The objectives of the proposed research are 1) to further assess the tumor producing capabilities of 7,12-dimethylbenz(a)anthracene (DMBA) induced nodule-like alveolar lesions (NLAL) and 2) to characterize the hyperactive outgrowths derived from enzymatically dissociated epithelial cells of DMBA treated glands in a serum-free, hormone supplemented organ culture of the whole mammary gland of BALB/c female mice. A limited number (2/12) of NLALs have been found to produce hyperactive lobuloalveolar (LA) outgrowths and one has so far produced a mammary adenocarcinoma. In order to obtain a more reliable assessment of the tumor potential of the NLAL, a larger sample of explants will be analyzed by the mammary fat pad transplantation procedure. Explants of NLAL cells in tissue fragments and enzymatically dissociated epithelial cells will be used. Several hyperactive LA outgrowths derived, one from a NLAL explant and 6 from explants of dissociated cells of DMBA treated glands are being carried by serial transplantation into mammary fat pads. Some of these outgrowths have produced mammary tumors. These outgrowth lines will be characterized for tumor potential and hormone sensitivity with respect to growth, and specific gene (casein) expression. Expression of the casein gene will be measured by molecular hybridization, using a specific complementary DNA to casein mRNA (cDNAcsn), synthesized in my laboratory. Hormone responses will be monitored in organ culture of the outgrowth in the inguinal mammary fat pad, cultivated in a hormone supplemented, chemically defined medium. Experiments have been outlined for testing two additional nonhydrocarbon, oncogens, N-methylnitrosourea (NMU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with respect to their potential to induce "transformation" of the epithelial and the stormal cells in culture of the whole mammary organ. The results should further the establishment of the usefulness of this singular culture model for studies on interactons among the carcinogens, hormones and different cell types under conditions of biological coexistence in the whole breast.